The role of increased cytosolic free calcium concentration, (Ca2+)I, in the development of lethal myocardial ischemic injury is unclear. It has been suggested that increased (Ca2+)I is a critical component in the pathogenesis of ischemic injury, but studies designed to test this hypothesis have been inconclusive because of the lack of a suitable methodology to measure (Ca2+)I changes in intact ischemic myocardium. The recent introduction of calcium and sodium indicators which can be accumlated in the cytoplasm of intact myocytes provides a method for measurement of (Ca2+)I. Recent studies of (Ca2+)I in the perfused rat heart using 19F NMR and 5FBAPTA, a fluorine-labelled calcium indicator, have shown that there is an increase in (Ca2+)I during ischemia, which occurs while the plasma membranes remain intact and is reversible upon reperfusion. Thus the initial increase in (Ca2+)I during ischemia is not a result of lethal injury but is a consequence of metabolic derangements in reversibly injured ischemic myocytes. The studies in this proposal are designed to determine whether the changes in (Ca2+)I contribute to the development of the structural manifestations of lethal injury and to determine how these two events are linked. The specific aims are 1) to evaluate the transport processes which regulate (Ca2+)I during energy deficient conditions with the goal of establishing protocols which result in different levels of (Ca2+)I during ischemia, 2) to evaluate the calcium-dependence of ultrastructural ischemic damage, 3) to evaluate the biochemical processes which are affected by increased (Ca2+)I and which could represent the link between increased (Ca2+)I and ultrastructural damage, and 4) to evaluate the effect of reperfusion conditions on (Ca2+)I during reperfusion and the relationship between (Ca2+)I during reperfusion and the development of lethal injury. This proposal relies on 19F NMR to measure changes in (Ca2+)I and (Na+)I in perfused rat hearts which contain fluorine-labelled calcium and/or sodium indicators. Hearts will be subjected to ischemia and anoxia. Perfusion conditions will be manipulated to enhance or suppress the rise in (Ca2+)I. Washout of fluorine- labelled indicators, enzyme release, immunofluorescence staining of cytoskeletal proteins involved in plasma membrane-to- cytoskeleton attachments, and electron microscopy will be evaluated to assess plasma membrane structural integrity. The proposed experiments will clarify whether there is a relationship between increased (Ca2+)I and ischemic injury and will suggest the nature of this relationship. These studies will also indicate the importance of derangements in calcium homeostasis in reperfusion injury following ischemia. The results could have important implications for design of therapeutic interventions to minimize infarct size during acute myocardial ischemia.